The immunoassay of the type which the invention is concerned is exemplified by an immunoperoxide assay for terminal deoxynucleotidyl transferase (TdT), a DNA polymerase enzyme found in lymphocyte cells of humans. Such an assay is marketed by the Coulter Immunology Division of Coulter Corporation, Hialeah, Florida which includes monoclonal antibody specific to TdT. TdT is particularly associated with T-cell differentiation. Elevated levels of the enzyme have been found to be symptomatic of certain human leukemias and has become a valuable marker for lymphoblastic neoplasms.
In performing a microscopic assay, a reliable control slide is desirable for maintaining the integrity of the analysis. Such a slide provides a standard against which to measure the sample cells being analyzed and assist the investigator in assuring that the reagents employed in preparing sample or specimen slides are functioning properly. Characteristic of such a control slide is the use of an intact cell specimen to serve as the control medium.
Significant obstacles have been encountered in preparing such a suitable control slide for TdT immunoassay because TdT is a fragile and sensitive enzyme, the expression of which is greatly affected by temperature, moisture and cell cycle. Heretofore, the control slide was prepared by "Cytospin" centrifuging approximately 5.times.10.sup.6 normal lymphocytic cells at approximately 1200 rpm for approximately eight minutes at a desired dilution of phosphate buffered saline (PBS) to form a monolayer of cells on a slide. The cell monolayer then was used in the assay. However, conventional processing alters the monolayer cell response in the assay. Shipment to the investigators and storage in the laboratory at proper temperatures were required. For instance, the control slides could be fixed in absolute methanol and shipped at a storage temperature of 4.degree. C. for up to 72 hours. Thereafter, the slides had to be stored at -70.degree. C. until shipped or needed for use by the laboratory. Shipment conditions could not be assured; many laboratories did not have a storage facility functioning at -70.degree. C.
Further experimentation with cytospin procedures established that "Cytospin" prepared slides could not be stored at 4.degree. C. or room temperature and maintain the ability to obtain positive staining for TdT. Shipment and storage at such optimistic temperature conditions would be desirable advantages for such a control slide where intact cells functioned as the control medium.
Providing such a TdT control slide stable at room temperatures for prolonged periods of time required consideration of three (3) variables which impact on the TdT in the cells, to wit, fixation, dehydration and heat. Recognized procedures such as lyophilizing or freeze-drying and paraffin embedding processing were considered. Lyophilizing might resolve moisture problems whereas paraffin embedding might resolve the exposure to heat problem, but no assured resolution of the detrimental effect of moisture on the cell line. Elimination of excess moisture is essential in order to obtain adequate TdT staining. However, excessive or improper dehydration impairs the ability to store a TdT control slide for prolonged periods of time at reduced temperatures. Of course, the morphology of the cells must be maintained in the control slide. All of these factors, as well as others, such as using formalin as the fixative, were to be considered where the cell line was to be used as the control medium.
It was known to use certain fixation compounds and paraffin-embedding where cell tissue was used as the control medium. However, these conventional techniques impacted adversely on a marker such as TdT enzyme which is very sensitive and fragile. For instance, formalin used as a fixative impaired the molecules within lymphocyte cells required to maintain adequate TdT staining for control use. Graded ethyl alcohol generally used for dehydration treatment of cells gave rise to excessive or improper dehydration. Prior art paraffin-embedding used high melting point paraffin (58.degree.-62.degree. C.) whereas such a technique impacted adversely on TdT cell line.
This invention succeeds in providing a control slide for an immunoassay of the TdT type wherein a lyophilized intact cell segment preferably is paraffin-embedded on a slide to function as the control medium. The control slide of the invention is substantially stable and non-degrading with respect to its authenticity at ordinary room temperature. The invention comprises also the method of making such an advantageous control slide.